Computational protocol: Maternal Vitamin C Deficiency during Pregnancy Persistently Impairs Hippocampal Neurogenesis in Offspring of Guinea Pigs

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Protocol publication

[…] 6 µm thick paraffin-embedded coronal sections were cut on a microtome (HM335E, Germany), placed on Superfrost® Plus Microscope slides and dried on a heating plate for 24 hrs at 40°C. Five consecutive sections were taken for every region each 400 µm apart, spanning the entire depth of the hippocampus (∼10 regions per animal). Cutting of the coronal sections was done with the guidance of the guinea pig brain atlas by Rapisarda and Bacchelli .Nissl staining was performed on the first section of every region after deparaffinization and rehydration. Stained sections were scanned with a Polaroid print scan 35plus and the hippocampal area was determined in a blinded fashion with ImageJ (Wayne Rasband, NIH). The hippocampal volume for each animal was then calculated by the Cavalieri principle, according to the formula : V = ∑ areas * distance between sections.To assess proliferation and migration of newborn cells in the hippocampus, immunofluorescence staining was performed as described previously on four out of eight coronal sections covering the depth of the DG, making each region 800 µm apart. Deparaffinized and rehydrated sections were boiled for 15 min in 10 mM sodium citrate buffer, pH 6.0 for optimized antigen retrieval of BrdU, nuclear DNA and cell markers . Sections were co-stained with polyclonal sheep anti-BrdU (Novus MB 500-235) and mouse monoclonal anti-NeuN (Chemicon MAB377) or mouse monoclonal anti-GFAP (Chemicon MAB360), and counterstained with 4′-6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei. The BrdU-positive cells in each part of the DG (hilus, subgranular zone [SGZ] and granular layer [GL]) were counted in a blinded fashion on stitched images taken at 20X magnification. Images were acquired on a wide-field epifluorescence microscope (AxioImager M1) using AxioVision Digital Imaging software (Carl Zeiss MicroImaging GmbH, Jena, Germany). [...] Statistical analysis of variance was performed on the collected data for animal body weight, brain weight, VitC concentration in brain and plasma, MWM performance, hippocampal volume, and number of BrdU positive cells in the different regions of the DG (Statistica v 7, Statsoft, Tulsa, OH, USA). Two-way ANOVA was applied to the stereological, immunohistochemical and biochemical data. In case of significance, Tukey’s post hoc test was applied. For analysis of MWM data, repeated measures ANOVA was applied to the cued test and acquisition phase of the non-cued MWM. Retention trial of the non-cued MWM was analyzed by one-way ANOVA. Comparison of swim-patterns was done with Fishers exact test. A P-value <0.05 was considered to be statistically significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Cavia porcellus
Diseases Ascorbic Acid Deficiency
Chemicals Ascorbic Acid, Bromodeoxyuridine